- Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells
The production of toxins by A. fumigatus may help the fungus to colonize and invade the respiratory epithelium by modifying the natural clearance of the respiratory tract. Previous research has shown that A. fumigatus culture filtrate modifies the transepithelial resistance (Rt) and transepithelial potential differences (Vt) of HNEC, an in vitro model of the air-liquid interface of airway epithelium . The aim of this study was to use HPLC and MS-MS to identify which toxins produced by A. fumigatus are responsible for these modifications. Our data suggest that verruculogen, which has never been implicated in invasive aspergillosis, is one of the probable candidates.
The fact that A. fumigatus produces a number of biologically active substances that slow ciliary beating, damage epithelium, and that may affect colonization of the airways has already been reported using culture explants . Among these substances, such toxins as gliotoxin, fumagillin, and helvolic acid have been implicated in the pathogenesis of aspergillosis . Our results did not suggest the involvement of any of these toxins in the effects observed on our in vitro model of respiratory epithelium. The HPLC fraction known to contain gliotoxin had no detectable effect, suggesting that the concentration of gliotoxin in the fungal filtrate was too low to play a role in the observed effects. For fumagillin, the effects were different from those of the whole A. fumigatus culture filtrate and were only observed with concentrations above those found in the culture filtrate. With the helvolic acid, we did not observe any effects at the tested concentrations based on the residual extract HPLC analysis. Therefore, we concluded that none of these metabolites were responsible for the electrophysiological modifications of HNEC. Nevertheless, their role, especially in association, cannot be completely ruled out. For instance, gliotoxin is known to be produced by A. fumigatus during the exponential growth phase at 37°C . Therefore, the role of gliotoxin may be minimal in the first days of colonization, after the seeding of airway epithelium, but become crucial at a later stage of infection. Gliotoxin has been detected in patients suffering from aspergillosis  and in bovine udder infected with A. fumigatus .
Our results strongly suggest that verruculogen is one of the candidate toxins, if not the sole toxin, responsible for the changes of HNEC observed with culture filtrate. Our arguments are based on the fact that: (i) only the fraction containing verruculogen modified Vt and Rt, (ii) the concentration of verruculogen in the organic extract corresponds to the concentration obtained from the standard curve produced with pure commercial toxin, (iii) the temperature of 25°C, which fosters the production of verruculogen by A. fumigatus, led to increased effects, and that (iv) heating the filtrate at 100°C for 10 min did not modify the effects on HNEC  and is known not to modify the structure of verruculogen (this was verified by HPLC analysis; not shown).
Verruculogen is one of the tremorgenic mycotoxins produced by fungi belonging to the genera Penicillium and Aspergillus that elicit intermittent or sustained tremors (staggers syndromes) in vertebrate animals [22,23]. The clinical symptoms typically observed during mycotoxin poisoning include diminished activity and immobility, followed by hyper-excitability, convulsions, muscle tremor, ataxia, and tetanic seizures. However, death rarely occurs and these symptoms are reversible if the animals are removed from the toxic-feed source. The concentrations detected in our A. fumigatus filtrates are considerably lower than those reported when clinical symptoms of poisoning are observed in ruminants. No data are yet available in relation to the concentration dependence of human susceptibility. Verruculogen is known as an inhibitor of K+ channels in synaptic in vitro models [23,24]. Since verruculogen has never been tested on HNEC, additional research is needed to identify the prime action site on HNEC. The action of verruculogen could be extended to other targets such as DNA .
In order to include verruculogen as one of the virulence factors of A. fumigatus, toxin should be present in all the A. fumigatus isolates. No specific isolates have been shown to be more pathogenic than others in humans [13,26,27] or birds . According to some authors, 40% to 91% of the A. fumigatus isolates produce verruculogen [28,29]. The production of mycotoxins at an easily detectable level is very dependent on culture medium and temperature. For instance, verruculogen is produced in larger amounts at 25°C than at 37°C . Generally, greater amounts of mycotoxins are detected in grain cultures than in standard liquid cultures. Moreover, the production of toxins on grains is more physiological for a mould than the production of toxins in microbiological medium such as PDA or Sabouraud medium. Using autoclaved grains as a culture support, we were able to show that 67 different A. fumigatus isolates are able to produce verruculogen. As we do not observe direct cell lysis with low concentrations of verruculogen, further studies are required to test whether verruculogen is a virulent factor in the strictest sense, i.e. a component that damages the host cells . However, a verruculogen-deficient mutant strain would probably not be informative, since the pathogenicity of A. fumigatus is multifactorial. Indeed, all experiments performed to prove the role of a specific molecule in the virulence of A. fumigatus have failed, with the exception of melanin-deficient mutants  and mutants deficient in the siderophore biosynthesis [31,32].
To take on a primary role in the colonization and/or invasion of airway epithelium, verruculogen not only has to be present in every A. fumigatus isolate, but also has to be associated with conidia and delivered directly onto the epithelial cells. The presence of verruculogen at the cellular level could alter pulmonary clearance and thereby increase colonization and the risk of invasive aspergillosis. Some authors have found verruculogen in very few conidial extracts . Using MS/MS, we found that verruculogen is associated with conidia (although contamination with residual hyphae is always possible). Therefore, verruculogen can act on its first contact with epithelial cells. Furthermore, the detection of verruculogen in conidial extracts presents opportunities for investigation into the potential role of inhalation in the intake of mycotoxins by humans. The recent detection of abundant respirable ergot alkaloids associated with A. fumigatus conidia raises the possibility of the respiratory epithelium as an alternative to the digestive route for the intake of these mycotoxins .
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